The major disadvantage with the therapeutic use of most biologicals is that they are administered through parenteral route e.g., intravenously (i.v.), subcutaneously (s.c.), intramuscularly (i.m.) etc., which means that delivery to the patient is associated with pain and discomfort. Further, because of their usually very short half-lives, biologicals require frequent administrations into the patient in order to maintain therapeutic blood levels of the drug. Many types of injections that cannot be self-administered, require frequent trips to the clinic, further adding to the discomfort of the patient. Multiple examples of such biological drugs that require frequent administration exist. Interferon alpha-2a (Roferon, Roche) and interferon alpha-2b (Intron A, Schering AG), the two recombinant forms of human interferon alpha, used in the treatment of chronic hepatitis B and C have a serum half-life of less than 12 h (McHutchison, et al., Engl. J. Med. 1998, 339, 1485-1492; Glue, et al., Clin. Pharmacol. Ther. 2000, 68, 556-567) and therefore requiring 3 times a week administration. Repeated injections with interferon beta-1b (Betaseron) are also required to treat the patients of multiple sclerosis (MS). The recommended dosing is by subcutaneous route given every other day. Another example of a drug where repeated injections are required is filgrastim (granulocyte colony stimulating factor, or G-CSF), where the injection is given everyday for the duration of treatment two weeks.
One very successful and well accepted method of overcoming the above drawbacks of frequent high dose injections to maintain threshold levels of the drug in the body is to increase the in-vivo half-life of the therapeutic protein by conjugating it with a polymer, preferably polyethylene glycol (PEG). PEG molecules with their long chains not only create a protective shield around the pegylated drug molecule in aqueous solution, thereby, reducing the immunogenicity of protein drugs while also protecting them from the action of proteases, but they further help increase circulation half-life of the drug by increasing its hydrodynamic volume which reduces its loss from the filtration mechanisms of the kidney glomeruli network. After their separation from the protein molecule, the PEG moieties are cleared without any structural changes and their clearance is proportional to their molecular weight. Conjugation of proteins to PEG has been reported since 1970s. Usually PEG moieties are attached to the protein by first activating the PEG moiety and then reacting it with the side chain of lysine residue and/or the N-terminal amino group on the protein. The most frequently used PEG is monofunctional PEG because this moiety resists cross-linking and aggregation. One such example has been disclosed by Davis et al. in U.S. Pat. No. 4,179,337. PEG_protein conjugates were formed by reacting a biologically active material with a molar excess concentration of a highly activated polymer having a terminal linking group without regard to where the polymer would attach to the protein, and leading to a physiologically active non-immunogenic water soluble polypeptide composition. Pegylation of interferons has been reported in U.S. Pat. Nos. 4,766,106 and 4,917,888 which describe inter alia beta interferon conjugated with activated polymers including mPEG-2,4,6-trichloro-S-triazine, mPEG-N-succinimidyl glutarate or mPEG-N-succinimidyl succinate. One such disclosure in U.S. Pat. No. 5,951,974 describes the conjugation of interferon to a substantially non-antigenic polymer at a histidine site. Another such disclosure in U.S. Pat. No. 5,981,709 describes the alpha interferon-polymer conjugate with relatively long circulating half-life in-vivo.
Some commercially available pegylated therapeutic proteins include, ADAGEN (pegylated bovine adenosine deaminase), which is used to treat X-linked severe combined immunogenicity syndrome; PEGASYS (pegylated alpha-interferon 2a), which is used in the treatment of hepatitis C; PEG-Intron (pegylated alpha-interferon 2b) for chronic hepatitis C; Oncaspar (pegylated L-asparaginase) for the treatment of acute lymphoblastic leukemia in patients who are hypersensitive to the native unmodified form of L-asparaginase; and, Neulasta (pegylated recombinant methionyl human granulocyte colony stimulating factor) for cancer chemotherapy induced neutropenia.
Hepatits C virus (HCV) is one of the major causes of liver disease in the world. Nearly 200 million people are affected world wide. Interferon in combination with ribavirin has been shown to be effective in decreasing the viral load of patients with chronic hepatitis C, however it needs to be given three times a week. PEG-interferon alpha 2b is a covalent conjugate of recombinant interferon alpha 2b with monomethoxy PEG in a 1:1 molar ratio (Glue P et al., Clin Pharmacol Ther. 2000; 68; 556-567). The mean absorption half-life of PEG-interferon alpha 2b is 5 fold greater than non-pegylated interferon alpha-2b. The mean elimination half-life is 40 hours in patients with hepatitis C infection. Another product is PEG-interferon alpha 2a, which has a 40 kDa branched chain molecule with each PEG branch with an average molecular weight of 20 kDa. The two monomethoxy PEG chains are joined via hydrolytically stable urethane bonds to a lysine linker molecule, one at the lysine alpha-amino group and another at the lysine ε-amino group. The mean absorption half-life of PEG-interferon alpha 2a is 10 fold greater than non-pegylated interferon alpha-2a. The mean elimination half-life is about 60 hours in patients with hepatitis C infection. Both these improved products need to be administered at once a week regimen only.
While, some protein-polymer conjugates are stable in the liquid form, others are not. For example, unlike the case of PEG-interferon alpha 2a where pegylation leads to a stable urethane bond, which is primarily stable in aqueous media, the PEG-interferon alpha 2b product, which contains PEG primarily linked to a histidine (His 34) residue, is highly unstable in the liquid form. With such protein-polymer conjugates, one has to use techniques such as lyophilization/freeze-drying—a process whereby water is sublimed from a composition after it is frozen—which can provide a stable form to the biological over a desired period of time. Thus, to make a stable formulation of PEG-interferon alpha 2b, one needs to carefully lyophilize the formulation with suitable cryoprotectant(s) or lyoprotectant(s), and stabilizers, that stabilize the pegylated interferon alpha conjugates to prevent depegylation during and after lyophilization—a phenomenon commonly associated with the PEG-interferon alpha 2b product. Further, besides the cryoprotectant and stabilizers the lyophilized formulation also contains bulking agents to increase the amount of the solid material in the vial.
One specific way in which the problem of instability of urethane linkage at His 34 residue has been resolved in the case of PEG-interferon alpha 2b, is by utilizing a formulation that has been disclosed in U.S. Pat. No. 6,180,096, where in the PEG-IFN alpha 2b conjugates are lyophilized in the presence of buffer, cryoprotectants, a stabilizer and a solvent of which one such formulation contains a disaccharide sucrose, as a cryoprotectant, along with, monobasic sodium phosphate dihydrate and dibasic sodium phosphate anhydrous, as buffer, with polysorbate 80 as a stabilizer, and water as a solvent. While the above formulation is commercially successful in the treatment of Hepatitis C, it is nevertheless associated with several problems some of which are elaborated in another patent application, WO2006/020720, by the same company, that sites longer lyophilization cycles leading to increased cost of manufacturing, and higher moisture content associated with the commercial formulation, as some of the reasons to discover and report novel formulation in WO2006/020720. In WO2006/020720, the inventors disclose another lyophilized formulation of PEG-IFN alpha 2b, wherein the cryoprotectant comprises of at least 60% trehalose, the buffering components comprise of monobasic sodium phosphate dihydrate and dibasic sodium phosphate anhydrous, and where the formulation further comprises of polysorbate 80 as a stabilizer and water as a solvent, and that is able to overcome the above described problems of the commercial formulation. The need for additional formulations for the protection of PEG-IFN alpha 2b conjugates cannot be better emphasized than the fact that the assignees of U.S. Pat. No. 6,180,096 (commercial formulation), and the applicants of WO2006/020720, are the same company, Schering Corporation, that is continuing to develop and disclose more lyophilized formulations for PEG-IFN alpha 2b.
The need for additional formulations of PEG-IFN alpha 2b to those in existence is with an aim not only to protect the PEG-interferon alpha conjugate during and after lyophilization, but also to have a long-term storage at room temperature when lyophilized in an appropriate container. The process of such formulations should be easy to handle and be more cost-effective than those used for the current formulation (sucrose based). The current commercial formulation of PEG-IFN alpha 2b, which is sucrose based (as described in U.S. Pat. No. 6,180,096), has a rather long lyophilization cycle of nearly 5 days. The formulation disclosed in the current invention uses a lyophilization cycle which is significantly shorter in time—approximately by 24-48 hours—which will help in significantly bringing down the cost of manufacturing this drug.
The present invention provides novel lyophilized and stabilized formulations of PEG-Interferon alpha conjugates and the process for their preparation.